Real life clinical outcomes of relapsed/refractory diffuse large B cell lymphoma in the rituximab era: The STRIDER study

Abstract Background Relapse and refractory (R/R) rates after first‐line R‐CHOP in diffuse large B cell lymphomas (DLBCL) are ~40% and ~15% respectively. Aims We conducted a retrospective real‐world analysis aimed at evaluating clinical outcomes of R/R DLBCL patients. Material and Methods Overall, 403 consecutive DLBCL patients treated in two large hematological centers in Torino, Italy were reviewed. Results At a median follow up of 50 months, 5‐year overall survival from diagnosis (OS‐1) was 66.5%, and 2‐year progression free survival (PFS‐1) was 68%. 134 (34.4%) patients relapsed (n = 46, 11.8%) or were refractory (n = 88, 22.6%) to R‐CHOP. Most employed salvage treatments included platinum salt‐based regimens in 38/134 (28.4%), lenalidomide in 14 (10.4%). Median OS and PFS after disease relapse or progression (OS‐2 and PFS‐2) were 6.7 and 5.1 months respectively. No significant difference in overall response rate, OS‐2 or PFS‐2 in patients treated with platinum‐based regimens versus other regimens was observed. By multivariate analysis, age between 60 and 80 years, germinal center B cell type cell of origin and extranodal involvement of <2 sites were associated with better OS‐2. Discussion Our findings confirm very poor outcomes of R/R DLBCL in the rituximab era. Widespread approval by national Medicine Agencies of novel treatments such as CAR‐T cells and bispecific antibodies as second‐line is eagerly awaited to improve these outcomes.


| INTRODUCTION
Clinical outcomes of patients with diffuse large B cell lymphoma (DLBCL) remain suboptimal with relapse and chemo-refractoriness rates up to 40%-50% and 10%-15% respectively. 1,24][5][6][7][8] The "classical" firstline treatment backbone, containing an anthracycline and an anti CD20 antibody (commonly "R-CHOP": rituximab, cyclophosphamide, vincristine, doxorubicin, prednisone), [9][10][11] has recently been challenged by the introduction of novel agents such as first-line polatuzumab vedotin, 12 while salvage treatment in the R/R setting has not yet been standardized, particularly for elderly patients. 1Even though second-line combinations with platinum salts and cytarabine, or etoposide, and rituximab (i.e., R-DHAP: rituximab, cytarabine, cisplatin and dexamethasone; R-OxDHA: rituximab, oxaliplatin, cytarabine and dexamethasone; R-ICE: rituximab, ifosfamide, etoposide, carboplatin; R-GDP: rituximab, gemcitabine, dexamethasone and cisplatin), followed by autologous stem cell transplantation (ASCT) as consolidation, are commonly employed for medically fit patients, [13][14][15][16] population-based studies report that over half of R/R patients undergo mainly palliation. 17For subsequent relapses, approved agents in Italy include single agent pixantrone and lenalidomide, with a median OS ranging between 8 and 10 months. 18,19Allogeneic stem cell transplantation (allo-SCT) is used only in selected cases with good response to re-induction therapy, with 4-year overall survival (OS) around 20%; its feasibility, however, is limited by patient age, comorbidities, and risk of treatmentrelated toxicities. 20,21cently, several novel strategies have been developed.However, their approval relies on national Medicine Agencies the policies of which differ significantly from country to country.The U.S. Food and Drug Administration (FDA) approved chimeric antigen receptor (CAR) T cell therapies axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel), in 2017 and 2018, respectively, for adult patients with high-grade B-cell lymphomas or DLBCL R/R to at least 2 therapy lines.In the ZUMA 1 and JULIET studies, overall response rate (ORR) were 82% and 52% with a complete response (CR) rate of 54% and 40% respectively. 22,23In the TRANSCEND study, 24 similar results were reported with lisocabtagene maraleucel, approved in 2021, and later extended also to refractory patients and to those relapsing within 12 months after first line treatment in June, 2022, according to the results of the TRANSFORM study. 25In long term safety and efficacy analyses median OS at 24 months was not reached for both axi-cel 26 and tisa-cel in patients who achieved CR at 3 and 6 months. 27ecently, axi-cel was FDA-approved for DLBCL refractory to first-line chemo-immunotherapy based on the ZUMA-7 study. 28Other recently approved agents include: tafasitamab, an Fc-enhanced, humanized, monoclonal antibody targeting CD19, used in combination with lenalidomide in adult patients not eligible for ASCT 29 ; the antibody-drug conjugate polatuzumab vedotin, a CD79bdirected antibody conjugated with monomethyl auristatin (MMAE), used in combination with bendamustine and rituximab after at least two prior therapies 30 ; loncastuximab tesirine, a CD19-directed antibody and alkylating agent conjugate 31 ; and the XPO inhibitor selinexor used as single agent after two to five systemic treatment lines. 32ere, we present a study designed to determine reallife clinical outcomes of R/R DLBCL in the rituximab era, and to possibly characterize baseline features at diagnosis that may predict poor response to first-line treatment and response to salvage therapies.analysis, age between 60 and 80 years, germinal center B cell type cell of origin and extranodal involvement of <2 sites were associated with better OS-2.

| Study design
The "STRIDER" ("strategies of treatment in diffuse large B cell lymphoma in the era of rituximab") is a retrospective, observational study designed to evaluate clinical outcomes of R/R DLBCL patients after first-line treatment in the rituximab era in a real-world setting.Between January 2010 and December 2019, patients older than 18 years, consecutively treated at 2 Tertiary Referral Centers (Division of Hematology-University of Torino, Italy, and Division of Hematology, AOU Città della Salute e della Scienza-Torino, Italy), were evaluated for enrollment.The study was proposed and discussed with the patients by the treating hematologist during follow up visits.Patients were enrolled after obtaining informed consent.The study was approved by the local Institutional Review Board (A.O.U.Città della Salute e della Scienza, Torino, Italy) and conducted according to the Declaration of Helsinki.Patient data were obtained from hospital health records and research files.All data were pseudo-anonymized by assignment of a study specific patient code.

| Inclusion criteria
Major inclusion criteria included initial biopsy-proven diagnosis of either DLBCL or high grade B cell lymphomas (HGBCL).Histological and immune-histochemical diagnosis by tru-cut core-needle biopsies was allowed, while cases diagnosed by fine needle aspiration cytology were excluded.Transformed disease after a previous diagnosis of low grade lymphoma was allowed.R/R disease was documented by biopsy, imaging studies or clinical evaluation; refractoriness to first line treatment was defined as reappearance or progression of DLBCL or HGBCL within 12 months from initial diagnosis or disease-related death (POD-12).For all patients, retrieved data included demographics, whole blood counts, basic metabolic panel; imaging studies (computed tomography, CT, and fluorodeoxyglucose-positron emission tomography, PET, if available); histology studies by lymph node biopsy, bone marrow biopsy and bone marrow aspirate; presence of B symptoms, performance status, prognostic scores [IPI, age-adjusted IPI, Central Nervous S,35ystem International Prognostic Index (CNS-IPI)]; number of therapy lines and regimens employed.Cell of origin (COO) was determined by Hans' algorithm method; bulky disease was defined as any lesion >6 cm by CT.

| Statistical analysis
Primary endpoint was OS for R/R patients after salvage treatment (OS-2).Secondary endpoints included progression free survival after first line treatment (PFS-1), PFS after salvage treatment defined as PFS-2; OS-1; POD12; determinants for survival outcomes; distribution of second line regimens (descriptive analysis); efficacy outcomes with salvage treatments (response and duration of response).Sample size estimation was not predefined, all consecutive patients meeting the inclusion criteria during the defined study period were eligible for the study though patients with missing data were excluded from the analysis.Response to therapy was determined by the 1999 International Working Group response criteria 33 and by Lugano response criteria for patients diagnosed after 2014. 34Baseline characteristics of R/R patients were compared to those of non R/R (NRR) patients to identify potential prognostic factors.Statistical analyses were carried out using R (v 4.3.1).Survival curves were plotted with Kaplan-Meier method and compared with log-rank test.Medians between groups for continuous variables were compared by the Kruskal-Wallis (for non-normal variables) or the one-way ANOVA test (for normal variables); the chi-squared test or Fisher's exact test for small study samples, were employed for categorical variables.The Cox proportional hazards model was implemented for the univariate and multivariate survival analyses.In particular, an AIC-based backward stepwise algorithm (R function stats::step) was used to perform the variable selection, from 12 initial variables (outcome after firstline, age, hemoglobin, COO, gender, ECOG performance status, stage, extranodal involvement, LDH, ki67, type of first-and second-line treatment) to 6 (age, hemoglobin, COO, extranodal involvement, ki67, type of second line treatment).This method allows to determine the most relevant covariates for the outcomes of interest and to analyze potential confounding factors for each covariate.As required by the algorithm, the dataset was restricted to the R/R patients for whom there were no missing data on the initial 12 variables (n = 48).Thus, multivariate analysis for OS-2 was re-run by applying a data imputation algorithm, the "Multiple Imputation by Chained Equations" (MICE), a robust, informative method to analyze datasets with missing data.The procedure "fills in" missing data through an iterative series of predictive models.In each iteration, a specified variable is imputed using the other variables in the dataset.These iterations are run until convergence has been met.POD12 was determined by logistic regression: the model predicts the probability of being refractory/relapsed/dead due to DLBCL within 12 months and the causing factors.
By multivariate analysis, in 48 R/R patients with a complete dataset, among 6 variables (age, hemoglobin, COO, extranodal involvement, ki67, type of second-line treatment), age between 60 and 80 years, GCB-type COO and extranodal involvement of <2 sites were significantly associated with OS-2 (Table 3).By implementing   on 55 patients with complete data (Table 3).Moreover, logistic regression was performed to highlight determinant features for early (<12 months) R/R disease (POD12), and significant factors were age older than 80 years (p = 0.005, OR 8.7) and stage III-IV (p = 0.011, OR 7.7, Table S2).Despite the significant improvement with the introduction of rituximab, clinical outcomes of patients with R/R DLBCL remain invariably poor.Recently, novel immunotherapies such as CAR T cells, antibody-drug conjugates (i.e., polatuzumab vedotin, loncastuximab tesirine), and bispecific antibodies (i.e., glofitamab, epcoritamab) have however shown impressive results.Nonetheless, these agents have not yet been largely approved as second-line therapies by many national Medicine Agencies.Thus, effective salvage treatments remain an urgent medical need.The STRIDER study is a large, retrospective, real life study that confirmed the dismal prognosis of R/R patients in the rituximab era.OS-2 was disappointingly short with rates of about 6 and 4 months in relapsed and refractory patients respectively, with no statistically significant differences between the 2 cohorts.The SCHOLAR-1, the largest pooled analysis on R/R DLBCL patients in the rituximab era, reported a median OS for refractory patients of 6.3 months from the start of salvage treatment. 115][16]35 Studies with DHAP and GDP reported 4-year OS of 39%, 35 and, with R-DHAP and R-ICE, 3-year OS of 49%. 14Though comparative studies were not designed, ORRs did not differ significantly ranging from 40% to 60%.R-GemOx, investigated in an older population with median age of 69 years, did not include consolidation with ASCT, 36 reporting 5-year OS of 14%.In our rituximab-exposed population, ORR with platinum-based regimens were similar to previous reports, though the initial relatively good ORR and CR rates did not translate into a survival advantage, likely due to short response duration.For this reason, many patients undergoing platinumbased regimens did not receive ASCT consolidation, thus explaining the similar results observed with other less intensive second-line salvage treatments.Of note, only 38/102 (37.3%) patients were however treated with platinum-containing regimens, showing that, in a real life setting, the administration of intensive salvage treatments is not feasible in most patients.Moreover, frailty and chemo-refractoriness, in our experience, were also documented by the fact that only 6 (15.8% of the candidates to transplant patients) receiving platinum-based schemes eventually underwent ASCT.In published reports, the "intent-to-salvage transplant" was as low as 30%. 14,35eliable clinical parameters predicting increased risk of refractoriness prior to R-CHOP are lacking.Given that every treatment cycle may reduce chemosensitivity, it would be important to identify factors that predict survival and response to salvage treatments.Previous studies showed that high-intermediate IPI score at relapse 37 and secondary aaIPI 38 affected response rate after salvage therapy while factors such as COO by Hans' algorithm at relapse, 39 relapse <12 months after initial therapy 40 and prior rituximab treatment 41 also affected prognosis.We tried to identify parameters, at diagnosis, that could predict inferior outcomes in case of relapse or progression.By multivariate analysis, age category, COO, and number of extranodal sites involved were predictive of OS-2.However, the current role of patient age should be re-assessed in the light of novel treatments with better toxicity profile compared to standard chemotherapy.Interestingly, our patients between 60 and 80 years showed a significant survival advantage over younger patients.This could partly be explained by more aggressive biologic features in younger patients that may be detected with novel molecular signatures.Of note, a prospective study by the Fondazione Italiana Linfomi (FIL) identified age over 80 years as an independent variable correlated with OS.Moreover, a novel prognostic score for elderly patients, the EPI score, by a simplified version of the geriatric assessment (sGA) was proposed.By classifying patients as fit, unfit, and frail, the EPI score risk correlated with OS of 75%, 58%, and 43%, respectively. 42As a matter of fact, at our centre, a thorough geriatric assessment is part of the initial clinical work up by which elderly patients are offered treatment based on their fitness.This may be the reason for the low rates of palliative care undergone by our patients at diagnosis (3.3%) and at relapse/refractoriness (20%) compared to other reports. 17ndeed, patients over 80 are frequently ineligible for chemotherapy due to comorbidities. 43Lenalidomide may be a valid option, especially in non-GCB patients, with reported ORRs ranging from 29% to 37% with up to 20% CR rates. 44,45We report ORR and CR of 64.3% and 50%, respectively, in 14 patients, median age 76 years, treated with second-line lenalidomide, whereas thirdline lenalidomide in 13 patients showed inferior outcomes.Finally, a recent real world experience reported outcomes and costs associated with CAR-T cell therapy in DLBCL patients older than 65 (of note; however, regulatory approval in Italy actually limit access to CAR-T programs only to patients <75 years old).Overall, median OS was 17.1 months with no difference between age groups (65-69; 70-74; >75 years). 46No patient in the STRIDER was treated with CAR-T cells as the study was conducted before CAR-T cell therapies were commercially approved after two treatment lines.
Splitting the COO category in GCB and non-GCB (NOS) patients by immunohistochemical analysis, [47][48][49] according to Hans' algorithm, evaluated at baseline in over half of our patients, identified a strong variable associated with both OS-1 and OS-2 by multivariate analysis.Overall, the role of COO, assessed even with more precise tools such as Nanostring platforms, has recently been questioned in favor of more complex gene signature classifications, able to detect DLBCL molecular heterogeneity and to predict clinical outcome.][7][8] With the emergence of new therapies, and the increasing biological understanding of DLBCL pathogenesis, efforts to re-design first-line treatments beyond R-CHOP are being made.A stringent baseline risk stratification and the availability of a dynamic risk evaluation in the follow up that includes first-line response and disease kinetics (i.e., early vs. late relapse) should allow to promptly identify poor prognosis patients, who may benefit from earlier interventions with novel immunotherapies, including CAR-T cells.

| CONCLUSIONS
The STRIDER is a large, retrospective, real life study that confirms the poor prognosis of R/R DLBCL patients in the rituximab era, before the implementation of "next generation" salvage treatments such as CAR-T cells and bispecific antibodies.Efficacy of high dose chemotherapy-based salvage treatments is limited, requiring the urgent and widespread approval of these novel immunotherapies, mainly investigated in clinical trials in Europe.

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Our findings confirm very poor outcomes of R/R DLBCL in the rituximab era.Widespread approval by national Medicine Agencies of novel treatments such as CAR-T cells and bispecific antibodies as second-line is eagerly awaited to improve these outcomes.K E Y W O R D S chemotherapy, diffuse large B cell lymphoma, real world, refractory disease, relapse, rituximab, transplantation 2 | MATERIALS AND METHODS

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I G U R E Overall survival (OS1, A) and progression free survival (PFS1, B) for non-relapsed/refractory patients, relapsed and refractory patients.F I G U R E Overall survival after first relapse/progression (OS2, A) and progression free survival after first relapse/progression (PFS2, B) from diagnosis.F I G U R E Overall survival (OS2, A) and progression free survival (PFS2, B) from second line treatment for relapsed and refractory patients by univariate analysis.the multiple imputation model for missing data of the 12 variables (n = 134), results did not significantly differ with age (p = 0.014), COO (p = 0.016) and number of extranodal sites (p = 0.034) remaining significantly associated with OS-2.No baseline feature resulted significantly associated with PFS-2 by multivariate analysis

F I G U R E 6
Overall survival (OS2) stratified by age (A), cell of origin (COO) (B) and by ECOG PS (C) at initial diagnosis by univariate analysis.F I G U R E 7 Overall survival (OS2, A) and progression free survival (PFS2, B) with platinum-based versus other regimens as second line treatment by univariate analysis.
Patient characteristics and treatments.